Tissue factor deficiency causes cardiac fibrosis and left ventricular dysfunction R. Pawlinski*, A. Fernandes*, B. Kehrle*, B. Pedersen*, G. Parry*, J. Erlich†, R. Pyo‡, D. Gutstein‡, J. Zhang‡, F. Castellinoʈ, E. Melis§, P. Carmeliet§, G. Baretton¶, T. Luther¶, M. Taubman‡, E. Rosenʈ, and N. Mackman*,** *Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037; †Department of Nephrology, University of New South Wales, Prince of Wales Hospital, High Street, Sydney 2052, Australia; ‡Department of Medicine and Molecular Biology, Mount Sinai Medical Center, One Gustave L. Levy Place, Box 1269, New York, NY 10029; §Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology (VIB), KU Leuven, B-3000 Leuven, Belgium; ¶Institut fu¨r Pathologie, Universita¨tsklinikum Carl Gustav Carus der Technischen Universita¨t, 01307 Dresden, Germany; and ʈDepartment of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556 Edited by Philip W. Majerus, Washington University School of Medicine, St. Louis, MO, and approved October 4, 2002 (received for review August 20, 2002) Exposure of blood to tissue factor (TF) activates the extrinsic Materials and Methods (TF:FVIIa) and intrinsic (FVIIIa:FIXa) pathways of coagulation. In this Mice. Low-TF mice (line 47) were analyzed on either a mixed Ϸ study, we found that mice expressing low levels of human TF ( 1% genetic background (62.5% C57BL͞6J, 25% 129Sv, and 12.5% ؊/؊ of wild-type levels) in an mTF background had significantly BALB͞c) or a C57BL͞6J background (Ն97%). mTF⌬cyt/⌬cyt shorter lifespans than wild-type mice, in part, because of sponta- mice express normal levels of murine TF lacking the cytoplasmic neous fatal hemorrhages. All low-TF mice exhibited a selective domain (17). Low murine FVII mice (FVIItTA-FVII/tTA-FVII) were heart defect that consisted of hemosiderin deposition and fibrosis. generated by replacing the entire FVII gene with a transgene Direct intracardiac measurement demonstrated a 30% reduction expressing FVII under the control of a tTA responsive promoter (P < 0.001) in left ventricular function in 8-month-old low-TF (E.R., Z. Liang, A. Martin, and F.C., unpublished data). FbgϪ/Ϫ mice compared with age-matched wild-type mice. Mice expressing Ϫ Ϫ and FIX / mice have been described (11, 18). All studies were low levels of murine FVII (Ϸ1% of wild-type levels) exhibited a approved by The Scripps Research Institute Animal Care and similar pattern of hemosiderin deposition and fibrosis in their Use Committee and comply with National Institutes of Health hearts. In contrast, FIX؊/؊ mice, a model of hemophilia B, had guidelines. normal hearts. Cardiac fibrosis in low-TF and low-FVII mice appears to be caused by hemorrhage from cardiac vessels due to impaired Measurement of Cell Counts and Clotting Activity. White cell, red hemostasis. We propose that TF expression by cardiac myocytes cell and platelet counts, and hemoglobin and hematocrit levels provides a secondary hemostatic barrier to protect the heart from were determined by LabCorp (San Diego), using blood collected hemorrhage. from the inferior vena cava. Activated partial thromboplastin times (APTTs) and prothrombin times (PTs) were performed xpression of tissue factor (TF) by adventitial fibroblasts and using Automated APTT Reagent and Thromboplastin Reagent BIOCHEMISTRY Evascular smooth muscle cells surrounding blood vessels (Organon Teknika), respectively, and clotting times determined provides a hemostatic barrier that activates coagulation when using a START4 Coagulation Analyzer (Diagnostica Stago, vascular integrity is disrupted (1). TF is also expressed by cardiac Parsippany, NJ). Levels of TAT in the plasma were determined muscle but not by skeletal muscle (1). TF functions as the ͞ using a commercial ELISA (Enzygnost, Dade Behring, Marburg, high-affinity cellular receptor for FVII VIIa (2). The coagula- Germany). The procoagulant activity of heart tissue extracts tion protease cascades are comprised of the extrinsic (TF:FVIIa) added to mouse plasma was determined using a one-stage and intrinsic (FVIIIa:FIXa) pathways, which together maintain clotting assay as described (13) and converted to activity units by hemostasis (3). comparison to a standard curve generated using mouse brain Many murine models of coagulation have been generated that thromboplastin. Factor VII assays were performed using a provide new insights into the role of the various procoagulant modification of the Coaset FVII assay (Chromogenix, Milan). A and anticoagulant proteins in hemostasis (4). For instance, standard curve was generated by combining wild-type mouse FVLeiden/Leiden mice, which express an FV variant that is resistant plasma with human FX-deficient plasma at varying ratios. to inactivation by activated protein C, and TMPro/Pro mice, which express a mutated version of thrombomodulin (TM) with re- Histology. Tissue sections were stained with hematoxylin͞eosin duced thrombin binding, both exhibit prothrombotic phenotypes (H&E), Prussian Blue, or Masson’s Trichrome. Macrophages with increased fibrin deposition in select tissues (5–7). Mice with were identified with a monoclonal antibody MOMA-2 (1:1,000; prohemorrhage phenotypes include models of hemophilia A (FVIIIϪ/Ϫ) and B (FIXϪ/Ϫ), as well as fibrinogen-deficient mice Serotec; ref. 20). (FbgϪ/Ϫ) and thrombocytopenic mice (NF-E2Ϫ/Ϫ) (8–12). Mice Measurement of Left Ventricular (LV) Function of the Hearts of Low-TF with complete deficiencies in TF, FVII, FX, FV, and prothrom- Mice. bin die in utero or shortly after birth (4). We and others have LV function of hearts of low-TF mice (8 months of age) and Ͻ age-matched C57BL͞6J mice was measured as described (20). generated mice expressing low levels ( 0.1–1% of wild-type ͞ levels) of human TF, murine FVII, and murine FV (13–15). We LV function (dp dt) and LV systolic pressures were obtained at have shown that low-TF mice have impaired uterine hemostasis a constant heart rate range of 480–510. The heart rate was (16). A similar phenotype is observed with low-FVII mice. decreased by increasing the level of isoflurane anesthesia. In this study, we performed a detailed characterization of low-TF mice. These mice exhibited shorter lifespans than wild- This paper was submitted directly (Track II) to the PNAS office. type mice. Histological analysis of various tissues of low-TF mice Abbreviations: Fbg, fibrinogen; H&E, hematoxylin͞eosin; LV, left ventricular; TF, tissue revealed hemosiderin deposition and fibrosis selectively in their factor. hearts. Our data suggest that cardiac fibrosis in low-TF mice is **To whom correspondence should be addressed at: Departments of Immunology and Cell caused by hemorrhage from cardiac vessels due to impaired Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, C-204, La Jolla, hemostasis. CA 92037. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.242501899 PNAS ͉ November 26, 2002 ͉ vol. 99 ͉ no. 24 ͉ 15333–15338 Downloaded by guest on September 30, 2021 Table 1. Clotting activity in low-TF mice Genotype mTFϩ/ϩ mTFϩ/Ϫ͞hTFϩ mTFϪ/Ϫ͞hTFϩ Heart PCA, arbitrary units 232 Ϯ 80 (3) 98 Ϯ 80 (3) 0.2 Ϯ 0.2 (3) PT, s 10.47 Ϯ 0.15 (3) 10.48 Ϯ 0.05 (4) 10.53 Ϯ 0.32 (4) APTT, s 26.37 Ϯ 0.90 (3) 29.85 Ϯ 0.76 (4) 31.53 Ϯ 0.90 (4) TAT, ng͞ml 1.78 Ϯ 0.44 (3) 0.74 Ϯ 0.28 (5) 0.04 Ϯ 0.02 (5) PT, prothrombin time; APTT, activated partial thromboplastin time; PCA, procoagulant activity. Numbers in parentheses indicate the number of mice used in each analysis. Data Analysis. Statistical analysis was performed using a two- and then became widespread throughout the myocardium tailed unpaired Student’s t test, and differences were determined (Fig. 2B). to be statistically significant at a P value of Ͻ0.05. Decreased LV Function in the Hearts of Low-TF Mice. Hemodynamic Results studies revealed that low-TF mice had a marked impairment of Characterization of Low-TF Mice. Low-TF mice (mTFϪ/Ϫ͞hTFϩ heart contractility manifested by a significant decrease in dp͞dt line 47) contain a minigene (hTF) that directs cell type-specific at every heart rate examined (Fig. 3). At normal mouse heart expression of human TF that is similar to the expression of rates (480–510 beats per min), the dp͞dt and the LV pressure murine TF (13). Immunohistochemical analysis indicated that were decreased by 30% (P Ͻ 0.001). These results indicate that human TF was expressed by adventitial cells surrounding blood the cardiac fibrosis in the hearts of low-TF mice significantly vessels (data not shown). However, quantitation of the proco- impairs LV function. agulant activity of tissue extracts from various tissues indicated that these mice expressed low levels of TF (Ϸ1% of wild-type Detailed Histological Analysis of the Hearts of Low-TF Mice. We levels; ref. 13). For example, the procoagulant activity of tissue examined hearts from Ͼ100 low-TF mice at different ages. We extracts from hearts of low-TF mice was very low compared with observed golden-brown granular deposits in the myocardium in control mice (Table 1), indicating a deficiency of TF in the heart. tissue sections stained with H&E (Fig. 4A). We suspected that We evaluated the clotting activity and response to hemostatic these deposits were hemosiderin, which is an insoluble protein challenge of low-TF mice (4–8 weeks of age) on a C57BL͞6J produced by phagocyte digestion of hematin. We used Prussian background. No significant differences were found in whole Blue staining to confirm the presence of iron in the deposits in blood samples collected from low-TF mice (n ϭ 5) and serial sections (Fig. 4B). Immunohistochemical studies showed ϩ Ϫ ϩ mTF / ͞hTF (n ϭ 5) littermate mice with regard to platelet, that the hemosiderin-laden cells stained with MOMA-2 (not red cell, and white cells counts, and hematocrit and hemoglobin shown), which specifically recognizes macrophages (19).